The secretion of penicillinase by Bacillus licheniformis and of invertase by the simple eukaryote yeast (Saccharomyces) are studied to clarify the mechanism of secretion in microorganisms and its relation to the process in animals cells. Membrane penicillinase, a phospholipoprotein, with an N-terminal phosphatidylserine, is an intermediate in formation of exoenzyme. The physiological role of the penicillinase-releasing enzyme (PRE) which cleaves membrane penicillinase will be assessed from its properties and specificity and the phenotype of mutants (ts) lacking PRE. The tendency of other phospholipoproteins to be secreted will be determined. Improvement of the in vitro protein synthesis system (B. licheniformis) will be designed to demonstrate when the N-terminal phosphatidylserine is added to the growing chain, if it is derived from the phosphatidylseryl tRNA of the plasma membrane, and if the mRNA codes for the repeating portion of the "tail". The subunit structure of the mannan protein form of invertase will be worked out by maleylating the enzyme and removing the mannan tufts enzymatically to decrease associative interactions. This should clarify the chemical relation between this enzyme and the carbohydrate-free internal form. The cell site (plasma membrane?) at which glycosylation occurs will be identified, and inhibitors employed to cause accumulation of intermediate forms. Tunicamycin, a specific inhibitor of glycoprotein biosynthesis, will be given special attention. The alkaline phosphatases (present in the vacuole) will be purified and their apparent carbohydrate portion characterized in order to learn why their synthesis is insensitive to tunicamycin (or 2-deoxy-D-glucose).